Step 1. After you have properly installed the column, conditioned it according to the suggestions supplied in the package, it is a
good idea to start with a gradient run. We suggest starting with an acidified mobile phase of water and
Acetonitrile. Acidify the mobile phase with up to 0.5% of Formic or Acetic Acid. If you are not
using LCMS, TFA is another good candidate.

Step 2. Run about 6 column volumes of the mobile phase in Step 1 at 95% Water.

Step 3. Set up your instrument to run a shallow gradient from 95% Water to 40% Water over 20 minutes on a
75mm long column. For longer columns increase the gradient time proportionally. This long and shallow gradient will
be very beneficial for determining the optimal gradient or isocratic method to run your mixture with later. For
sharper peaks and less retention, run a shorter (Steeper) gradient from the same starting points to end points.

Step 4. Equilibrate the column by running 100% Acetonitrile for approximately 2 minutes.

Step 5. Set up your instrument to run a shallow gradient using the same mobile phase in Step 3 to run from
90% Acetonitrile to 40% Acetonitrile over 20 minutes for a 75mm long column. For longer columns increase the
gradient time proportionally. This long and shallow gradient will be very beneficial for determining the optimal
gradient or isocratic method to run your mixture with later. For sharper peaks and less retention, run a shorter
(Steeper) gradient from the same starting points to end points.

Step 6. Evaluate both gradient runs for retention time, peak shape and elution order as retention on the
column is compound specific where some compounds will not retain in Step 3 (Reverse Phase) and some do not retain
in Step 5 (Aqueous Normal Phase). One column could produce an isocratic run which retains both polar and non polar
compounds.

Note: The Cogent Bidentate C18 columns have a unique quality in that they can retain
non polar compounds not retained on other columns even when others run at 100% water. You could insert an isocratic run at 100%
Water after Step 3 and before Step 4 without loss of precision.